The present invention relates to genes and the proteins encoded thereby which are involved in neurotoxicity and/or are regulated by FK506. Polynucleotides were discovered using in vivo or in vitro models by determining which genes were differentially upregulated or downregulated when subjected to various stresses, such as hypoxia, and/or upon treatment of the model with FK506. Polynucleotides were also found by a functional selection (assay) of cDNA fragments specifically selected for their ability to confer cell resistance to various stresses which can result in neurotoxicity, such as hypoxia, glutamate or dopamine treatment. The invention includes such polynucleotides, corresponding genes, and proteins encoded thereby, as well as naturally-occurring variants of such polynucleotides, analogs, salts and functional derivatives of such proteins, DNA encoding such analogs, antibodies, antisense molecules and methods of use. Such methods of use include methods for protecting cells from neurotoxicity and ameliorating the effects of stroke, hypoxia and/or ischemia by regulating such genes or proteins.
Brain injury such as trauma and stroke are among the leading causes of mortality and disability in the modern world.
Traumatic brain injury (TBI) is one of the most serious reasons for hospital admission and disability in modern society. Clinical experience suggests that TBI may be classified into primary damage occurring immediately after injury, and secondary damage, which occurs during several days post injury. Current therapy of TBI is either surgical or else mainly symptomatic. Stroke is the third leading cause of death and disability in developed countries, affecting more than half a million Americans each year. Stroke is an acute neurologic injury occurring as a result of an insult to the brain, thus interrupting its blood supply. Stroke induces neuronal cell death, which leads to the clinical outcomes of patients"" death or disability ranging from total paralysis to milder dysfunction. Cerebral ischemia is the most common type of stroke, which may lead to irreversible neuronal damage at the core of the ischemic focus, whereas neuronal dysfunction in the penumbra may be reversible. Cells in the penumbra have an estimated time window for survival of up to 6 hours. The ability to intervene as soon as the patient is identified is essential for recovery. It is well established that ischemic tissue damage is multifactorial and involves at least excitotoxicity, reactive oxygen species, and inflammationxe2x80x94all leading to neuronal cell death.
Treatment strategies for stroke are aimed to induce rapid reperfusion and rescue of neurons in the penumbral area. Neuroprotective drugs are constantly being developed in an effort to rescue neurons in the penumbra from dying. However, potential cerebroprotective agents need to counteract all the above-mentioned destructive mechanisms. Therefore, current therapy in stroke focuses primarily on prevention, minimizing subsequent worsening of the infarction, and decreasing edema.
FK506 (tacrolimus) is a known immunosuppressive agent produced by Streptomyces tsukubaesis, a species discovered by the Fujisawa Pharmaceuticals"" scientists in a soil sample from Tsukuba, Japan. See Kino et al, 1987, and U.S. Pat. No. 5,338,684. FK506 possesses neuroprotective activity by delaying or preventing hypoxia-induced death of neuronal cells. In addition, it can cause regrowth of damaged nerve cells. The specific molecular mechanism underlying the neuroprotective activity of FK506 is largely unknown although there are indications for suppression of activities of calcineurin and nitric oxide synthase as well as prevention of stroke induced generation of ceramide and Fas signaling. An additional model has been proposed involving steroid receptor complexes in context of FK506 neurotrophic actions. As a first step to novel drug discovery, these mechanisms should be delineated and key genes involved in these processes should be identified.
The polynucleotides of the present invention have been discovered by merging two technologies:
(1) microarray-based differential gene expression, evaluated in both in viva and in vitro models, and
(2) direct functional selection of genes with pro- or anti-apoptotic activities, performed in cell systems subjected to neurotoxic stress, such as hypoxia, glutamate or dopamine.
Differential profiling of gene expression was performed both in an in vivo model of permanent ischemia in rats either treated or untreated with FK506, performed by electrocoagulation of middle cerebral artery (MCA), and in an in vitro model of primary rat cerebellar neuron cultures exposed to hypoxia, with or without FK506 treatment. Polynucleotides were identified which were either upregulated or downregulated by either ischemia/hypoxia or the FK506 treatment or influenced by the combination of both treatments. Two proprietary cDNA microarrays, the xe2x80x9cApoptosisxe2x80x9d and xe2x80x9cStrokexe2x80x9d chips, were used in this study.
In addition, a direct functional selection of genes exhibiting pro-or anti-apoptotic activities induced by hypoxia, glutamate or dopamine was done on BE2C, an established human neuroblastoma cell line, upon introduction of expression cDNA library cloned into retroviral vector.
Accordingly, the present invention is directed to either novel polynucleotides whose expression (or function) in cells, in particular neural cells is modulated when cells are subjected to neurotoxic stress or whose activity is important for transduction of neurotoxic signals.
A total of 131 fragments, SEQ ID Nos: 1-131, were characterized as polynucleotides located in genes whose expression in neural cells is modulated when cells are subjected to neurotoxic stress or whose activity is important for neurotoxic signal transduction. Of these, 14 fragments which are incorporated in cDNA clones (all being KIAA clones) have been identified in our selection procedures as particularly preferred. This includes all of the polynucleotides of SEQ ID NOs:49, 50, 51, 65, 67, 85, 87 and 94-100, as well as the naturally-occurring full-length RNAs and corresponding full-length cDNAs and genes and natural antisense polynucleotides which include any one of these sequences, and corresponding polypeptides and proteins encoded by them.
Currently most preferred according to the present invention are the polynucleotides identified as SEQ ID NO: 94, which is a fragment of KIAA 0538 and SEQ ID NO: 65 which is a fragment of KIAA 0284. The former of these has been further identified as encoding a Ca2+-dependent Ras-GTPase Activator Protein. Elevated expression of Ras-GAP results in increased Ras inactivation and may contribute to cell death, in particular neuronal cell death.
The invention is further directed to naturally-occurring polynucleotides having at least 70% identity with any of the polynucleotides which include any one of SEQ ID Nos: 1-131, preferably SEQ ID Nos: 49, 50, 51, 65, 67, 85, 87 and 94-100, or which are capable of hybridizing under moderately stringent conditions to any of such polynucleotides, and whose expression or activity in naturally-occurring neural cells is modulated when the cells are subjected to neurotoxic stress.
The present invention is also directed to the polynucleotide comprising the sequence of any one of SEQ ID Nos: 1-48, 52-64, 66, 68-84, 86, 88-93, 101-131, which are novel polynucleotides and genes. The expression or activity of these polynucleotides in naturally-occurring neural cells is modulated when the cells are subjected to neurotoxic stress.
The present invention is also directed to fragments having at least 20 nucleotides of any of the polynucleotides of the present invention and to polynucleotide sequences complementary to any of such polynucleotides or fragment and to polypeptides encoded by any of the polynucleotides of the present invention.
In a more preferred embodiment, the isolated polynucleotide is a strand of a full-length cDNA.
According to one currently more preferred embodiment, the invention particularly encompasses methods for screening drugs which upregulate or downregulate a gene which is transcribed to an RNA containing a sequence of any of SEQ ID Nos: 1-131, preferably SEQ ID NOs: 49, 50, 51, 65, 67, 85, 87 and 94-100.
According to another more preferred embodiment, the present invention provides methods for screening a compound which induces or inhibits apoptosis after exposure of neural cells or other cells such as glia, lymphocytes, macrophages to a neurotoxic insult.
According to yet another more preferred embodiment, the present invention provides methods of screening for a compound capable of exerting a neuroprotective effect that ameliorates or diminishes the damage induced by a neurotoxic insult.
The present invention is further directed to isolated proteins or polypeptides encoded by any such full-length cDNA, as well as variants which have an amino acid sequence having at least 70% identity to such an isolated protein and retain the biological activity thereof, or biologically active fragments of such protein or variant, as well as to salts or functional derivatives of any such protein, variant or biologically active fragment. The expression or activity of these polypeptides in naturally-occurring neural cells is modulated when the cells are subjected to neurotoxic stress. The present invention is preferably directed to the polypeptides encoded by polynucleotides comprising the sequence of any one of SEQ ID Nos: 1-48, 52-64, 66, 68-84, 86, 88-93, 101-131.
The present invention is also directed to antibodies specific to any of the proteins, variants or fragments of the present invention and to any molecule comprising the antigen-binding portion of any such antibody, in particular to the antibodies. The present invention is particularly directed to antibodies specific to the polypeptides encoded by polynucleotides comprising the sequence of any one of SEQ ID Nos: 1-48, 52-64, 66, 68-84, 86, 88-93, 101-131, which are novel polypeptides. The present invention is also directed to a molecule which comprises the antigen-binding portion of an antibody specific for a protein, variant or fragment.
The present invention also comprehends antisense DNA/RNA of a length sufficient to prevent transcription and/or translation of any gene identified in accordance with the present invention, preferably comprising a sequence which is complementary to a portion of a gene of which a sequence of SEQ ID NO:94 is a part or complementary to a portion of a gene of the KIAA0538 gene family. The present invention also comprehends ribozymes which specifically bind and cleave mRNA sequences identified in accordance with the present invention.
The present invention further comprehends methods of treating the effects of stroke, hypoxia and/or ischemia, and neurotoxicity as well as for diagnosing cells which have been subjected to hypoxia and/or ischemia, using the polynucleotides, polypeptides/proteins, antibodies, or ribozymes of the present invention.
The present invention further comprehends methods of treating the effects of neurotoxicity, stroke, hypoxia, or ischemia, comprising regulating in the cells to be treated the level of expression of any of the polynucleotides of the present invention, preferably polynucleotides comprising SEQ ID Nos: 49, 50, 51, 65, 67, 85, 87, 94-100, most preferably polynucleotides comprising SEQ ID No: 94 (corresponding to KIAA0538).
The present invention further comprehends methods of treating the effects of neurotoxicity, stroke, hypoxia, or ischemia, comprising bringing into the vicinity of the cells to be treated any of the polypeptides of the invention, preferably a polypeptide encoded by a cDNA comprising a polynucleotide having a sequence SEQ ID NO:65 (corresponding to KIAA0284), a variant which has an amino acid sequence having at least 70% identity to said polypeptide and retains the biological activity thereof, or a fragment of said polypeptide or variant which retains the biological activity thereof, or a functional derivative or salt of said protein, variant or fragment.
The present invention further comprehends methods of treating the effects of neurotoxicity, stroke, hypoxia, and/or ischemia, comprising bringing into the vicinity of the cells to be treated an agent for inhibiting the polypeptide a protein encoded by a cDNA selected from SEQ ID No:94 or KIAA0538, a variant which has an amino acid sequence having at least 70% identity to said protein and retains the biological activity thereof, or a fragment of said protein or variant which retains the biological activity thereof, or a functional derivative or salt of said protein, variant or fragment.
The present invention further comprehends methods for the treatment of a subject in need of treatment for the effects of neurotoxicity, stroke, hypoxia, or ischemia, comprising regulating in said subject the level of expression of any of the polynucleotides of the invention , so as to thereby treat the subject, preferably preferably polynucleotides comprising SEQ ID Nos: 49, 50, 51, 65, 67, 85, 87, 94-100, most preferably polynucleotides comprising SEQ ID No: 94 (corresponding to KIAA0538).
The present invention further comprehends methods for the treatment of a subject in need of treatment for the effects of neurotoxicity, stroke, hypoxia, or ischemia, comprising administering to said subject a polypeptide encoded by a cDNA selected from SEQ ID NO:65 or KIAA0284, a variant which has an amino acid sequence having at least 70% identity to said protein and retains the biological activity thereof, or a fragment of said protein or variant which retains the biological activity thereof, or a functional derivative or salt of said protein, variant or fragment.
The present invention further comprehends, as a preferred embodiment, the treatment of a subject in need of treatment for the effects of neurotoxicity, stroke, hypoxia, or ischemia, comprising administering to said subject a compound for inhibiting a polypeptide comprising a protein encoded by a cDNA selected from SEQ ID No:94 or KIAA0538, a variant which has an amino acid sequence having at least 70% identity to said protein and retains the biological activity thereof, or a fragment of said protein or variant which retains the biological activity thereof, or a functional derivative or salt of said protein, variant or fragment.
The present invention further comprehends methods for diagnosing cells which have been subjected to a neurotoxic insult, hypoxia and/or ischemia, comprising assaying for RNA comprising a sequence of any one of SEQ 1-131, preferably RNA comprising a sequence of any one of SEQ ID NOs:49, 50, 51, 65, 67, 85, 87, 94-100, most preferably RNA comprising a sequence of any one of SEQ ID NOs: 65, or 94, or for the expression product of a gene in which one of said sequences is a part, the change in amount of said RNA or expression product as compared to a control indicating the likelihood that such cells have been subjected to hypoxia or ischemia.
The present invention further comprehends methods of screening for a neuroprotective compound comprising testing the ability of the compound to upregulate or downregulate a gene which is transcribed to an RNA containing a sequence of any of SEQ ID NOs: 1-131, preferably any of SEQ ID NOs: 49, 50, 51, 65, 67, 85, 87 and 94-100, most preferably downregulate the transcription of SEQ ID No:94 or KIAA0538 or upregulate the transcription of SEQ ID No65 or KIAA 0284.
The present invention further comprehends methods of identifying a neuroprotective compound or screening for a neuroprotective compound comprising testing the ability of the compound to inhibit or enhance the activity of a polypeptide which is encoded by a polynucleotide of any of SEQ ID NOs: 1-131, as compared to a control, preferably SEQ ID NOs: 49, 50, 51, 65, 67, 85, 87 and 94-100, most preferably to inhibit SEQ ID NO 94, even more preferably wherein the compound is screened for the ability to inhibit a Ca2+ promoted Ras inactivator encoded by a member of the KIAA0538 gene family; another preferred embodiment is wherein the compound is screened for the ability to activate or enhance the activity of a polypeptide encoded by KIAA0284.
The present invention further comprehends methods for screening for a compound or identifying a compound which induces or inhibits apoptosis after exposure of mammalian cells, preferably neural cells, to a neurotoxic insult, comprising the step of exposing the cells to the test compound and testing the change in expression, as compared to a control, of any one of the polynucleotides of the invention, preferably the change in expression of any member of the KIAA0538 gene family or the expression of KIAA0284.
The present invention further comprehends methods for screening for a compound or identifying a compound which induces or inhibits apoptosis after exposure of mammalian cells, preferably neural cells, to a neurotoxic insult, comprising the step of exposing the cells to the test compound and testing the change in activity of any one of the polypeptides of the invention , as compared to a control, preferably where the compound is screened for the ability to inhibit a Ca2+ promoted Ras inactivator encoded by a member of the KIAA0538 gene family.
The present invention further comprehends methods for screening for a compound or identifying a compound capable of exerting a neuroprotective effect that ameliorates or diminishes the damage induced by a neurotoxic insult, comprising the step of screening for the ability of the compound to alter the level of expression of any of the polynucleotides of the invention, compared to a control, testing the compound for its ability to inhibit the expression of any polynucleotide of the KIAA0538 gene family.
The present invention further comprehends methods for screening for a compound or identifying a compound capable of exerting a neuroprotective effect that ameliorates or diminishes the damage induced by a neurotoxic insult, comprising the step of screening for the ability of the compound to alter the activity by enhancement or inhibition of any one of the polypeptides of the invention, preferably wherein the compound is screened for its ability to inhibit the activity of a Ca2+ promoted Ras inactivator encoded by a member of the KIAA0538 gene family.
The present invention further comprehends methods for screening for a compound or identifying a compound which up-regulate or downregulate a gene, the improvement wherein said gene is a gene which is transcribed to an RNA complementary to any of the polynucleotides of the invention.
The present invention further comprehends methods for screening for or identifying a neuroprotective compound which specifically inhibits the polypeptide product of KIAA0538 gene which comprises:
(a) contacting cells expressing DNA encoding the KIAA0538 gene under conditions permitting expression of the DNA; and
(b) determining if the compound inhibits the polypeptide as compared to a control; preferably the cells in this method are either transfected with the KIAA0538 gene or endogenously express the KIAA0538 gene, most preferably the cells are neuronal cells.
The present invention further comprehends a method of preparing a pharmaceutical composition which comprises the steps of:
(a) obtaining a compound which specifically inhibits the activity of the polypeptide product of the KIAA0538 gene; and
(b) admixing said compound with a pharmaceutically acceptable carrier. The inhibitory compound may be obtained by using one of the screening assay methods disclosed herein for identifying such compounds.
The compositions and methods of invention can be used to treat the adverse consequences of central nervous system injuries that result from any of a variety of conditions. Thrombus, embolus, and systemic hypotension are among the most common causes of cerebral ischemic episodes. Other injuries may be caused by hypertension, hypertensive cerebral vascular disease, rupture of an aneurysm, an angioma, blood dyscrasias, cardiac failure, cardiac arrest, cardiogenic shock, septic shock, head trauma, spinal cord trauma, seizure, bleeding from tumor, or other blood loss. Where the ischemia is associated with stroke, it can be either global or focal ischemia, as defined below.
The present invention is additionally directed to pharmaceutical compositions which include the nucleic acids, proteins or polypeptides in accordance with the present invention, along with pharmaceutically acceptable carriers or excipients.
In addition, the present invention is directed to knockout or transgenic non-human animals, in which a gene identified by the present invention has been introduced or knocked out.